title: Membrane cholesterol and sphingomyelin, and ostreolysin A are obligatory for pore-formation by a MACPF/CDC-like pore-forming protein, pleurotolysin B.
abstract: The mushroom Pleurotus ostreatus has been reported to produce the hemolytic proteins ostreolysin (OlyA), pleurotolysin A (PlyA) and pleurotolysin B (PlyB). The present study of the native and recombinant proteins dissects out their lipid-binding characteristics and their roles in lipid binding and membrane permeabilization. Using lipid-binding studies, permeabilization of erythrocytes, large unilamellar vesicles of various lipid compositions, and electron microscopy, we show that OlyA, a PlyA homolog, preferentially binds to membranes rich in sterol and sphingomyelin, but it does not permeabilize them. The N-terminally truncated Δ48PlyB corresponds to the mature and active form of native PlyB, and it has a membrane attack complex-perforin (MACPF) domain. Δ48PlyB spontaneously oligomerizes in solution, and binds weakly to various lipid membranes but is not able to perforate them. However, binding of Δ48PlyB to the cholesterol and sphingomyelin membranes, and consequently, their permeabilization is dramatically promoted in the presence of OlyA. On these membranes, Δ48PlyB and OlyA form predominantly 13-meric oligomers. These are rosette-like structures with a thickness of ∼9 nm from the membrane surface, with 19.7 nm and 4.9 nm outer and inner diameters, respectively. When present on opposing vesicle membranes, these oligomers can dimerize and thus promote aggregation of vesicles. Based on the structural and functional characteristics of Δ48PlyB, we suggest that it shares some features with MACPF/cholesterol-dependent cytolysin (CDC) proteins. OlyA is obligatory for the Δ48PlyB permeabilization of membranes rich in cholesterol and sphingomyelin.
authors.affiliation: Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, SI-1000 Ljubljana, Slovenia.
authors.forename: Katja
authors.initials: K
authors.lastname: Ota
authors.forename: Adrijana
authors.initials: A
authors.lastname: Leonardi
authors.forename: Miha
authors.initials: M
authors.lastname: Mikelj
authors.forename: Matej
authors.initials: M
authors.lastname: Skočaj
authors.forename: Therese
authors.initials: T
authors.lastname: Wohlschlager
authors.forename: Markus
authors.initials: M
authors.lastname: Künzler
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
authors.forename: Mojca
authors.initials: M
authors.lastname: Narat
authors.forename: Igor
authors.initials: I
authors.lastname: Križaj
authors.forename: Gregor
authors.initials: G
authors.lastname: Anderluh
authors.forename: Kristina
authors.initials: K
authors.lastname: Sepčić
authors.forename: Peter
authors.initials: P
authors.lastname: Maček
mesh_terms: D000818:Animals; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects; D002784:Cholesterol / Q000737:chemistry*; D004912:Erythrocytes / Q000166:cytology / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D006461:Hemolysis / Q000187:drug effects; D021962:Membrane Microdomains / Q000737:chemistry; D008854:Microscopy, Electron; D020076:Pleurotus / Q000737:chemistry*; D052899:Pore Forming Cytotoxic Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D013109:Sphingomyelins / Q000737:chemistry*; D053835:Unilamellar Liposomes / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D006460:Hemolysin Proteins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins; D013109:Sphingomyelins; D053835:Unilamellar Liposomes; C461280:ostreolysin; C586632:pleurotolysin B, oyster mushroom; D002784:Cholesterol
keywords: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Aegerolysin; C-terminally hexa-histidine tagged OlyA6; CDC; Cholesterol-dependent cytolysin; H(6)-OlyA6; H(6)-Δ48PlyB; Lipid-binding protein; Membrane attack complex; N-terminally hexa-histidine-tagged N-truncated precursor of PlyB (Acc. code AB177872, lacking 48 amino acids at N-terminus); N-terminally hexa-histidine-tagged OlyA6; OlyA6; OlyA6-H(6); POPC; Pleurotolysin; Pleurotus ostreatus; cholesterol-dependent cytolysin; nOlyA; nPlyB; native ostreolysin A; native pleurotolysin B; ostreolysin A6